Method and kit for predicting cancer

ABSTRACT

The present invention relates to a method and a kit for diagnosing and/or predicting the occurrence of cancer or the risk of contracting a cancer by measuring the concentration of a cancer screening antigen (CSA) in blood, which changes before the occurrence of the cancer in a patient. The method of diagnosing or predicting the occurrence of cancer or the risk of contracting a cancer comprising the steps of: determining a concentration of galectin-3 in a blood sample by reacting the blood sample with a monoclonal antibody of the galectin-3; comparing the determined concentration of the galectin-3 with concentration of the galectin-3 in a blood sample of a normal human; and predicting the risk of contracting a cancer if the determined concentration is greater than the concentration of the galectin-3 in blood of the normal human.

FIELD OF THE INVENTION

The present invention relates to a method and a kit for predictingcancer in a simple and easy manner. More particularly, the presentinvention relates to a method and a kit for predicting and/or diagnosingthe occurrence of cancer or the risk of contracting a cancer bymeasuring the concentration of a cancer screening antigen (CSA) inblood, which changes before the occurrence of the cancer in a patient.The present invention is particularly useful for (1) a patient having acancer, but no subjective symptom, (2) a patient having a benign tumor,a chronic inflammation or gastritis, but no subjective symptom, and (3)a normal person. The patients in the state of above (1) and (2) arecapable of predicting the occurrence of cancer or the risk ofcontracting a cancer with the kit and method according to the presentinvention, and may have a more precise medical examination, which helpsdiagnosis of the cancer in early stage.

BACKGROUNDS OF THE INVENTION

The detection of a cancer in early stage is most important in healingthe cancer. In most case, the subjective symptom due to a cancer isrevealed after the cancer is complicated. The medical check upexamination for cancer in hospital is generally not easy for ordinaryperson, and this makes it difficult to find out the cancer in earlystage. There is a continuing need for a method to predict the occurrenceof cancer or the risk of contracting a cancer in a simple and easymanner so that ordinary person can self-examine the occurrence of cancerconveniently and in early stage of the cancer development.

To develop simple and easy method to diagnose and remedy cancer,immunologists have tried to find out tumor-specific transplantationantigens (TSTA). However, due to the characteristic fact that the tumoris derived from the normal cell of a human, the TSTA is not identifiedexcept for some malignant epithelioma. Meanwhile, it is also tried touse tumor-associated transplantation antigens (TATA) such as analpha-fetoprotein (AFP) and a carcinoembryogenic antigen (CEA), whichexists in normal cell, but increases in tumor cell, for diagnosing thecancer. However, the over-manifestation of TATA occurs only after thetumor is complicated. Therefore, the method for diagnosing tumor withTATA is not much helpful in early diagnosing the cancer.

Galectins is a β-galactoside-binding lectin, and exists in most plantand animal including a poriferan, an eelworm, mammamlia, etc, and hashigh affinity to a laminin which is an extracellular matrix (ECM) ofanimal, and known to have close relation with the metastasis of tumor[26, 27]. In addition, it is also known that the galectins have closerelation with the growth and transformation of a cell, and metastasis ofa tumor. Until now, nine kinds of galectin are identified, and form agalectin family. The galectins have molecular weight of 14-36 kDa, andexists in monomer or dimer forms. The galectin-1, galectin-3, andgalectin-8 exist in various internal organs, and galectin-2, galectin-4,galectin-5 and galectin-7 exist in specific internal organs or cells [2,10, 15, 17, 25].

Galectin-3 is also called as CBP-35, Mac-2, ε BP, RL-29, L-34, L-31 etc,and discovered by Ho and Springer [12] in the macrophage activated bythioglycollate, and is a protein having molecular weight of about 26-32kDa [27]. The galectin-3 relates with the growth, differentiation,malignant degeneration, and embryo formation of a cell, and also relateswith the hypersensitivity reaction of a cell mediated with IgE, andplays important role in the binding between cells, and between cells andmatrix. The galectin-3 accelerates the uptake of the calcium ion inhuman Jurkat T-cell [8], and has relation with the apoptosis of a T-cell[29]. Galectin-3 can exist in the form of ribonucleoprotein(RNP)/galectin-3 complex, and effects the pre-mRNA substrate and the RNAsplicing process [7].

It is also known that the galectin-3 can be released from a cell withoutsignal peptide [31]. As the biological materials having the samereleasing property, interleukin-1 (IL-1) and fibroblast growth factor(FGF) are known, but the precise releasing mechanism is not identifiedyet. This releasing property is quietly different form that of generalsoluble protein in eukaryotic cell.

The galectin-3 protein is known to have close relation with malignanttumor, and occurs in colon cancer, skin cancer, thyroid gland cancer,breast carcinoma etc [5, 6, 9, 14, 18, 19, 20, 23, 24, 28]. There aremuch less research in the field of stomach cancer than colon cancer,lung cancer, breast carcinoma and malignant melanoma. The galectin-3does not manifest in the normal liver cell, but manifests in livercancer and cardiac cirrhosis [13]. In case of the thyroid gland cancer,it is known that the manifestation of the galectin-3 can be used as amarker before cancer operation [20]. In case of stomach cancer, it isreported that the galectin-3 does not manifest in normal stomach tissue,but manifests in the stomach cancer [30]. In summary, the heretoforeresearches indicate that the manifestation of galectin-3 has closerelation with the malignant tumor. However, the precise relation ofgalectin-3 with the tumor development, especially with stomach cancerdevelopment, is not established. Furthermore, the use of the galectin-3in predicting and/or diagnosing the occurrence of cancer or the risk ofcontracting cancer is not known.

SUMMARY OF THE INVENTION

In view of the foregoing, I identified the relation of manifestation ofgaletin-3 and development of tumor, and discovered theover-manifestation of galetin-3 in the stage of a benign tumor or achronic inflammation and gastritis, which is a former or an initialstage of tumor development. Thus, the present invention is directed tothe simple and easy method of using the over-manifestation of galetin-3in the former and initial stage of tumor development.

Therefore, it is an object of the present invention to provide a methodand a kit for diagnosing and/or predicting the occurrence of cancer orthe risk of contracting a cancer in the former and initial stage oftumor development by measuring the concentration of galectin-3 in blood,which is over-manifested in the stage of a benign tumor (adenoma) or achronic inflammation which is the former or initial stage of themalignant tumor (carcinoma). The user recognizing the occurrence ofcancer or the risk of contracting a cancer with the kit and methodaccording to the present invention may have a more precise medicalexamination in hospitals, which results in the early and exact diagnosisof the cancer.

In other words, the kit and method according to the present inventionmay help the user to self-examine his or her risk of contracting acancer in the initial stage of tumor development, and contributes to theearly finding of cancer, especially the stomach cancer, in the earlystage of tumor development. Therefore, it is other object of the presentinvention to provide a method and a kit for diagnosing and/or predictingthe occurrence of cancer of user having no subjective symptom.

To accomplish these and other advantages, the present invention providesa method of diagnosing and/or predicting the occurrence of a cancer orthe risk of contracting a cancer comprising the steps of: determining aconcentration of galectin-3 in a blood sample by reacting the bloodsample with a monoclonal antibody of the galectin-3; comparing thedetermined concentration of the galectin-3 with concentration of thegalectin-3 in a blood sample of a normal human; and predicting the riskof contracting a cancer if the determined concentration is greater thanthe concentration of the galectin-3 in blood of the normal human.

The present invention also provides a method of diagnosing or predictingthe occurrence of cancer or the risk of contracting a cancer comprisingthe steps of: preparing an assay strip having a reaction part, a sampleinjection part, and a membrane for providing passage from the sampleinjection part to the reaction part, wherein a capture antibody ofgalectin-3 or galectin-3 is immobilized on the reaction part; moving ablood sample including galectin-3 and a gold-conjugated tracer antibodyof the galectin-3 trough the membrane from the sample injection part tothe reaction part; and predicting the risk of contracting a canceraccording to the color change of the reaction part.

The present invention further provides a kit for diagnosing orpredicting the occurrence of cancer or the risk of contracting a cancercomprising an assay strip having a reaction part, a sample injectionpart, and a membrane for providing sample passage from the sampleinjection part to the reaction part, wherein the reaction part includesa capture antibody of galectin-3 or galectin-3 immobilized thereon sothat a color of the reaction part is determined according to theconcentration of galectin-3 in a blood sample when the blood sampleincluding galectin-3 and a gold-conjugated tracer antibody of thegalectin-3 reaches from the sample injection part to the reaction parttrough the membrane.

The present invention further provides a kit for diagnosing orpredicting the occurrence of cancer or the risk of contracting a cancercomprising: a microplate for immobilizing galectin-3 in a blood sample;and a monoclonal antibody to react with the galectin-3 immobilized onthe microplate to induce a color change of the microplate.

The present invention further provides a method of diagnosing orpredicting the occurrence of cancer or the risk of contracting a cancercomprising the steps of: determining a cancer screening antigen whichmanifests in the stage of a adenoma or a chronic inflammation which arethe former stages of the malignant tumor development; determining aconcentration of the cancer screening antigen in a blood sample byreacting the blood sample with a monoclonal antibody of the cancerscreening antigen; comparing the determined concentration of the cancerscreening antigen with a concentration of the cancer screening antigenin a blood sample of a normal human; and predicting the risk ofcontracting a cancer if the determined concentration is substantiallygreater or lesser than the concentration of the cancer screening antigenin blood of the normal human.

BRIEF DESCRIPTION OF THE DRAWINGS & PHOTOGRAPHS

A more complete appreciation of the invention, and many of the attendantadvantages thereof, will be readily apparent as the same becomes betterunderstood by reference to the following detailed description whenconsidered in conjunction with the accompanying drawings andphotographs, wherein:

FIG. 1A is a microscope photograph showing the manifestation ofgalectins-3 at Auerbach plexus surrounded by normal cell (magnifyingratio: 200);

FIG. 1B is a microscope photograph showing the manifestation ofgalectins-3 at semi-differentiated signet ring cell (magnifying ratio:200);

FIG. 2A is a microscope photograph showing the manifestation ofgalectins-3 at intestinal metaplasia (magnifying ratio: 100);

FIG. 2B is a microscope photograph showing the manifestation ofgalectins-3 at tubular carcinoma (magnifying ratio: 200);

FIG. 3 is a microscope photograph showing the manifestation ofgalectins-3 at well-differentiated tumor tissue and semi-differentiatedsignet ring cell (magnifying ratio: 200);

FIG. 4 is a graph showing the ratio of 5 years survival according to thedegree of the manifestation of galectins-3; and

FIG. 5 is a graph showing the concentration of galectins-3 in blood ofnormal human, patient having stomach cancer, and patient having chronicgastritis. The concentrations of galectins-3 in blood sample, which isdiluted successively, were represented by absorbance measured by theELISA method. In FIG. 5, x-axis represents dilution ratio of the bloodsample, and y-axis represents absorbance at 490 nm. Each mark in FIG. 5is depicted to represent mean±standard deviation.

FIG. 6 is a plan view of an assay strip forming the kit according to anembodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

For a better understanding of the present invention, reference will nowbe made in detail to the following disclosures and appended claims.

The present invention provides a method for diagnosing and/or predictingthe occurrence of cancer or the risk of contracting a cancer, such asstomach cancer, liver cancer, thyroid gland cancer etc, at the former orinitial stage of tumor development. Heretofore, the cancer is diagnosedby detecting the concentration of the tumor-associated transplantationantigens (TATA), which exists in normal cell, but increases in tumorcell. However, the over-manifestation of TATA occurs when the tumor iscomplicated, i.e. after the initial stage of the tumor development.Therefore, the method for diagnosing tumor with TATA is not much helpfulin early diagnosing the cancer.

In contrast, the present invention is directed to a new concept forearly diagnosing the tumor development. The method according to thepresent invention is to diagnose and/or predict the risk of contractinga cancer with an antigen which is over-manifested in blood before thedevelopment of the malignant tumor (carcinoma), rather than using anantigen which is over-manifested after the development of the carcinoma.The antigen, which is used in the present invention, is defined as acancer screening antigen (CSA).

The present invention also provide a kit utilizing the characteristicsof the cancer screening antigen (CSA) so that an ordinary user candiagnose and/or predict the occurrence of a cancer such as stomachcancer, liver cancer, thyroid gland cancer etc, preferably the stomachcancer, in early stage of the cancer development and in simple and easymanner. According to the present invention, the suitable cancerscreening antigen is the galectin-3. The galectin-3 is not manifested innormal tissue such as normal stomach tissue, liver tissue and thyroidgland, but over-manifested in the stage of a benign tumor (adenoma) or achronic inflammation which is the former stage of the malignant tumor(carcinoma). Thus, the galectin-3 can be used for early diagnosing thestomach cancer. In addition, since the liver cancer and thyroid glandcancer have same characteristics with the stomach cancer, the galectin-3can be used for early diagnosing the liver cancer and the thyroid glandcancer.

The method of using the galectin-3 in predicting the occurrence of atumor such as stomach cancer includes the steps of obtaining a bloodsample of a user, determining the concentration of the galectin-3 in theblood sample via antigen-antibody reaction, and comparing the determinedconcentration of the galectin-3 with the concentration of the galectin-3in a blood of a normal human who is not in the stage of a benign tumor(adenoma), a chronic inflammation, or the malignant tumor (carcinoma).The galectin-3 is not manifested in normal blood, but over-manifested inthe stage of the adenoma or the chronic inflammation, which is theformer stage of the carcinoma, and the over-manifestation is maintainedin the stage of the carcinoma. Thus, if the concentration of thegalectin-3 in the blood sample of a user is greater than that of thenormal human, the user might be in the initial or former stage of thedisease, and there is high probability of contracting the tumor.

Because the method according to the present invention diagnoses cancerby determining the concentration of the galectin-3 in blood samplerather than directly examining the tissues of stomach, liver and thyroidgland, the discrimination of the specific type of cancer, such asstomach cancer, liver cancer and thyroid gland cancer, cannot becomplete. However, if the test result is positive, the user may have amore precise medical examination, which results in the early and exactdiagnosis of the cancer.

The first step of diagnosing tumor according to the method of thepresent invention is to determine the concentration of the galectin-3 inthe blood sample by reacting the blood sample with a monoclonal antibodyof the galectin-3. The concentration of the galectin-3 in the bloodsample can be determined by an immunological test method, preferably byan enzyme-immunological test method which uses the antigen-antibodyreaction.

In the case of using an enzyme-linked Immunosorbent assay (ELISA), whichis one of the enzyme-immunological test methods, the antigen(galectin-3) in a blood sample is immobilized onto a stationary phase.For example, the antigen is coated on the stationary phase such as amicroplate. Preferably, the blood serum sample is diluted with a buffersolution before the immobilization with the dilution ratio of 32-256.After immobilizing the antigen, the stationary phase can be washed andblocked.

Then a monoclonal antibody (the 1st antibody of the galectin-3, forexample, M3/38 monoclonal antibody (rat IgG)) is added to the stationaryphase to react with the antigen, and washed, and then the 2nd antibody(for example, a horse radish peroxidase (HRP)-conjugated goat anti-ratIgG) is added to react with the 1st antibody which is also an antigen ofthe 2nd antibody. Preferably, the 2nd antibody is marked with enzyme.After the 2nd antibody marked with enzyme is combined to the 1stantibody, a colorant solution is added to the 2nd antibody to induceenzyme reaction and clear color change.

After stopping the enzyme reaction, the light absorbance of thegallectin-3 and the monoclonal antibody complex is measured to determinethe concentration of the galectin-3 in the blood sample. Since the 1stantibody, the 2nd antibody, enzyme, and the colorant are generally wellknown in the art, the selection thereof can be easily carried out byskilled person in the art, and preferably only the 1st antibody markedwith enzyme can be used without the 2nd antibody.

Alternatively, a kit such as an Immunochromatographic assay strip, whichhas similar configuration with the conventional kits for detectingpregnancy, diabetes or cholesterol content in blood, can be used fordetermining the concentration of the galectin-3.

Such kit may have various configurations which are well known in theart. As shown in FIG. 6, the simplest configuration of the kit is anassay strip 100 which includes a reaction part 10, a sample injectionpart 20, and a membrane 30 for providing passage from the sampleinjection part 20 to the reaction part 10. The assay strip 100 can beprotected by a plastic case having at least one window for observing thereaction part 10 and for injecting the sample.

In one embodiment, a capture antibody of the galectin-3 is immobilizedto the reaction part 10, and a blood sample including the antigen(galectin-3) and gold-conjugated tracer antibody of the galectin-3 aremoved trough the membrane 30 from the sample injection part 20 to thereaction part 10. In this embodiment if the galectin-3 isover-manifested in the blood sample, the more galectin-3 reacts with thegold-conjugated tracer antibody, and the greater amount of theantibody-antigen complex is captured on the reaction part 10, whichresults in the color change of the reaction part 10. On the contrary, ifthe galectin-3 is not over-manifested in the blood sample, thegold-conjugated tracer antibody cannot react with the antigen, thuscannot be captured on the reaction part 10. In this case, the color ofthe reaction part 10 cannot be changed. Therefore, a user can determinethe concentration of the galectin-3 according to the color change of thereaction part 10.

The gold-conjugated tracer antibody can be directly mixed with the bloodsample and injected onto the sample injection part 20. Alternatively,the gold-conjugated tracer antibody can be positioned between the sampleinjection part 20 and the reaction part 10 in a dried state. In lattercase, the dried gold-conjugated tracer antibody melts when the bloodsample moves from the sample injection part 20 to the reaction part 10,and reacts with the galectin-3 in the blood sample.

In other embodiment, galectin-3 is immobilized to the reaction part 10,and a blood sample including galectin-3 and gold-conjugated tracerantibody of the galectin-3 are moved trough the membrane 30 from thesample injection part 20 to the reaction part 10. When the blood sampleincluding the galectin-3 and gold-conjugated tracer antibody reaches thereaction part 10, the galectin-3 immobilized onto the reaction part 10and the galectin-3 in the blood sample competitively react with thegold-conjugated tracer antibody. Therefore, if the galectin-3 isover-manifested in the blood sample, the galectin-3 immobilized onto thereaction part 10 cannot react with the gold-conjugated tracer antibody.Thus, the color of the reaction part 10 cannot be changed. On thecontrary, if the galectin-3 is not over-manifested in the blood sample,the galectin-3 immobilized onto the reaction part 10 captures largeamount of the gold-conjugated tracer antibody, which results in thecolor change of the reaction part 10.

In summary, the reaction part 10 includes the capture antibody ofgalectin-3 or galectin-3 immobilized thereon so that the color of thereaction part 10 is determined according to the concentration ofgalectin-3 in the blood sample when the blood sample includinggalectin-3 and the gold-conjugated tracer antibody of the galectin-3reaches to the reaction part 10 trough the membrane 30.

To clearly detect the color change of the reaction part 10, the bloodsample can be decolorized according to the conventional method, such asmixing the blood sample with an oxidant. Alternatively, a filter forfiltering the coloring matter in the blood sample can be formed betweenthe sample injection part 20 and the reaction part 10. Preferably, aconventional control line 40 can be formed on the membrane 30 at theopposite end of the sample injection part 20, and the control line 40 isdesigned to change its color when the blood sample arrives to thecontrol line 40, which makes the user to confirm the test is completed.

Generally, even the normal tissue includes small amount of galectin-3.Therefore, the color change of the kit should be controlled so that thecolor change should occur when the concentration of the galectin-3 inthe blood sample is greater than that in the normal blood. In addition,the color change due to the concentration of the galectin-3 depends onthe dilution ratio of the sample (See following Example). Therefore, thesample dilution ratio should be controlled so that the color of thereaction part cannot be changed with the normal blood, and can bechanged with the abnormal blood. The dilution ratio can be properlycontrolled according to the kit configuration. In addition, anyconventional kit configuration disclosed in U.S. Pat. No. 5,616,467 canbe used as a kit configuration of the present invention.

In order to more fully illustrate the preferred embodiments of thepresent invention, the following detailed examples are given.

Example

1. Material and method.

(1). Preparation of Stomach Cancer Tissue and Blood

100 stomach cancer tissue slides were obtained from the Aju UniversityHospital in Republic of Korea. The tissue slides were collected duringthe gastrectomy operations of the 100 patients who were diagnosed tohave stomach cancer by pathological diagnosis during 1994-1996. Thetissue slides consisted of tissues under advanced carcinomas, earlygastric carcinomas (EGC), precancerous lesions, and normal gastricmucosal tissues. The normal peripheral blood were obtained from normalperson who are selected in random, and the peripheral blood of patientshaving stomach cancer or gastritis were obtained from the Aju UniversityHospital and Catholic University Vincent Hospital in Republic of Korea.

(2). Immunohistochemical Staining of the Galectin-3 Protein in StomachCancer tissue

In order to remove the paraffin in the prepared slides and to hydratethe slides, the tissue slides were dipped into xylene solution for 5minutes, and the dipping process was repeated 3 times. Then the slideswere successively dipped into 100% ethanol solution, other 100% ethanolsolution, 90% ethanol solution, 80% ethanol solution, and 70% ethanolsolution each for 5 minutes. Then the slides were dipped into distilledwater for 10 minutes, and 3% hydrogen peroxide solution for 5 minutes,and washed with PBS containing 1% Triton X-100, 3 times each for 5minutes. The washed slides were moved into a humidified chamber in orderto prevent non-specific binding reactions, and treated with 10% normalgoat serum, and reacted for 30 minutes at room temperature.

After the reaction was complete, the culture supernatant of cell strainproducing M3/38 monoclonal antibody (rat IgG, 1st antibody) was added tothe humidified chamber, and maintained at 4° C. for over-night, andwashed with PBS containing 1% Triton X-100 3 times each for 5 minutes.As the 2nd antibody, a horse radish peroxidase (HRP)-conjugated goatanti-rat IgG (Pierce co.) was diluted with PBS containing 3% BSA withthe dilution ratio of 1:800, and was added to the humidified chamber,and reacted for 1 hour at room temperature, and washed with PBScontaining 1% Triton X-100 3 times each for 5 minutes. Then the tissueis reacted with 3,3′-diaminobenzidine (DAB), and washed, and stainedwith Harris' hematoxylin as control, and dehydrated.

(3). Measurement of Concentration of the Galectin-3 in Peripheral Blood

The concentration of the galectin-3 in peripheral blood was measured asfollows with ELISA (Enzyme-linked Immunosorbent Assay).

100 μl of buffer solution including same amount of 0.5M carbonate and0.5M bicarbonate (pH=9.6) was coated on two 96 well plates,respectively. Then, 50 μl of the buffer solution and 50 μl of the bloodsample including antigen (galectin-3) was coated onto one of the 96 wellplates. The mixture was mixed well, and 100 μl of the mixture wastransferred to the 2nd 96-well plate to dilute the sample 2 times. Byrepeating the dilution process, the mixture was diluted to 2048 times,and in some experiment, diluted to 131072 times.

The diluted mixtures were maintained at 4° C. for over-night to coat theantigen (galectin-3) onto the 96-well plate. Then the coated antigen waswashed with washing solution (PBS+0.05% Tween-20) 3 times, and blockedwith PSB containing 3% BSA, and cultured for 1 hour at 37° C. Then themicroplate was washed.

Then, the 1st antigen was added to the coated antigen. In detail, theprepared monoclonal antibody of the galectin-3 was diluted with EIAbuffer solution having the following composition with the dilution ratioof 1:1, and inoculated to the well (100 μl/well), and incubated for 2hours at 37° C., and washed. < Composition of EIA buffer solution (100ml) > 0.01M tris-HCl buffer solution 75 ml Tween-20 0.1 ml Fetal bovineserum 25 ml EDTA 200 mg Thimerosal 5 mg

Then the 2nd antibody was reacted. In detail, goat anti rat Ig G-HRP wasdiluted with the dilution ratio of 1:1,500, and inoculated to the well(100 μl/well), and incubated for 2 hours at 37° C., and washed. The nextstep was to inoculate chromogen solution to the well (100 μl/well). Atthis time, a stock solution was prepared so that the final concentrationof OPD was 0.5 mg/ml, and the buffer solution of the OPD substrateconsisted of 0.05M sodium citrate and 0.05M Na₂HPO₄ (pH 5.0). The HRPsubstrate was prepared by mixing 1 ml of OPD stock(ortho-phenylenediamine stock solution), 9 ml of OPD buffer solution,and 3.3 μl of 30% H₂O₂, and the mixture was incubated for 1 hour at roomtemperature. Then the reaction was stopped by inoculation of 2.5M H₂SO₄(50 μl/well), and the light absorbance of the plate at 490 nm wasmeasured.

(4). Pathological Interpretation

The manifestation of galectin-3 at Auerbach plexus disposed in stomachwas regarded as control. The pathological diagnosis was interpretedaccording to the following criteria.

-   -   (−): galectin-3 is not manifested    -   (1+): galectin-3 is manifested in the tumor cell of less than 5%    -   (2+): galectin-3 is manifested in the tumor cell of 5-50%    -   (3+): galectin-3 is manifested in the tumor cell of more than        50%.

(5). Grouping and Statistical Analysis

The four criteria are divided into 2 groups as follows for comparing thepathological parameters according to the manifestation of thegalectin-3.

-   -   A group: 3+    -   B group: −, 1+, 2+.

Pathological parameters, such as a depth of invasion, number of lymphnode metastasis, and TNM stage according to the UICC classificationmethod between the two groups were analyzed with the Chi-square testmethod, and the ratios of survival of the two groups according to themanifestation of the galectin-3 was determined with Kaplan-Meier method.When P value was less than 0.05, the data is regarded as statisticallyuseful.

2. Result

(1). Immunohistochemical Staining

From the manifestation of the galectin-3 at Auerbach plexus (See FIGS.1A and 1B), it is clear that galectin-3 was seldom manifested in normalcell, and only small amount of the galectin-3 was manifested at themucosal neck portion. As shown in FIGS. 2A and 2B, the galectin-3 ismore strongly manifested in the stage of intestinal metaplasia andadenoma which are former stages of the tumor. As shown in FIG. 3, thegalectin-3 is widely manifested in the tumor tissue, but the degree ofthe manifestation increases with the degree of differentiation of acell. Thus the degree of the manifestation at the stage of thedifferentiation of a cell is greater than that of the stage of themalignant tumor.

(2). Comparison of the Two Groups According to the Degree ofManifestation of the Galectin-3

100 patients having stomach cancer included 72 men and 28 women, andtheir average age was 53.7 (age range: 27-78). Among them, 64 peoplewere classified into the group A, and 36 people were classified intogroup B. The pathological parameters of the two groups were compared,and the results are shown in Table 1. In case of group B, the numbers ofintestinal type and diffuse type are almost same, but in case of groupA, the number of intestinal type was greater than that of the diffusetype (p<0.05). In case of the depth of invasion, the ratio of T3 was thegreatest in both of the two groups (p<0.05), and the number of lymphnode metastasis was in the range of 1-6(p<0.05).

The ratios of stage III among stage I-IV were greatest, but the p-valuewas greater than 0.05, and the test results were regarded as not usefulstatistically. TABLE 1 Pathological analysis of the patient groupsaccording to the manifestation of the galactin-3. Group A Group BPathological parameter Number (%) Number (%) P-value Lauren IntestinalType 52(81.3) 17(47.2) <0.05 Diffuse Type 12(18.8) 19(52.8) Depth ofInvasion T1  8(12.5)  3(8.3) <0.05 T2  6(9.4)  7(19.4) T3 50(78.1)23(63.9) T4  0(0)  3(8.3) No. of Positive LNs 0 17(26.6)  3(8.3) <0.051-6 22(34.4) 20(55.6) 7-15 14(21.9) 10(27.8) more than 16 11(17.2) 3(8.3) Stage I  8(12.5)  3(8.3) 0.648 II 16(25.0)  6(16.7) III 29(45.3)20(55.6) IV 11(17.2)  7(19.4)The degree of manifestation of galectin-3 of Group A(n = 64): 3+The degree of manifestation of galectin-3 in Group B(n = 36): −, 1+, 2+

As shown in FIG. 4 showing the ratio of 5 years survival according tothe manifestation of the galectin-3, the ratio of 5 years survival ofthe Group A was 64.06%, and the ratio of 5 years survival of the Group Bwas 63.89% (p value=0.9153). Thus, it is found that the manifestation ofthe galectin-3 has no relation with the expected life time of thepatients.

(3). Detection of the Galectin-3 at Peripheral Blood.

The peripheral blood was obtained from the groups of normal human,patient having chronic gastritis, patient having benign tumor, andpatient having stomach cancer, respectively. The concentrations ofgalectin-3 were measured, and the results are depicted in FIG. 5. Asshown in FIG. 5, the manifestations of the galectin-3 of the groups ofchronic gastritis, benign tumor, and stomach cancer were greater thanthat of normal human. Thus, the galectin-3 can be used more effectivelyas a cancer anticipating or predicting antigen rather than cancerdiagnosing agent. The discriminating power of the manifestations wasmaximized with 100 times dilution of the blood sample.

3. Discussion

From the above-described experiment, the degree of manifestation of thegalectin-3 at each stage of the tumor development was determined, andits relation with the tumor developments was discovered. In sum, thegalectin-3 seldom manifests in normal cell, but strongly manifests inthe stage of intestinal metaplasia and adenoma which are the formerstages of the tumor. The most unexpected result obtained from theexperiment is that the galectin-3 more strongly manifests in the stageof intestinal metaplasia and adenoma than in the stage of the tumor. Themanifestation of the galectin-3 is more stronger in the welldifferentiated tumor cell than in undifferentiated tumor cell. Thismeans that the manifestation of the galectin-3 increases with theinitiation of intestinal metaplasia and adenoma, and is maximized duringthe stages of intestinal metaplasia and adenoma, and maintained when theintestinal metaplasia and adenoma are developed to tumor.

From the pathological parameter analysis, the manifestation of thegalectin-3 had little relation with the depth of invasion and the numberof lymph node metastasis. This result is similar to the result ofresearch of breast carcinoma, and different from the result of researchof colon cancer, lung cancer, melanoma etc, in which the manifestationof the galectin-3 has close relation with development of malignanttumor. From the experiment to the stomach cancer, it is found that themanifestation of the galectin-3 has little relation with the ratio ofsurvival of patients (p=0.9153), which means that the galectin-3 is notso effective as a diagnostic marker specific to the malignant tumor.

However, in the present invention, the galectin-3 is mainly used as adiagnosing agent of the intestinal metaplasia and adenoma rather thanthe malignant tumor. The galectin-3 is expected to have a role in theinitial or middle stages of the development of carcinogenesis since thegalectin-3 has mainly manifested at the former or initial stage of thedevelopment of the stomach cancer. Therefore, the manifestation of thegalectin-3 is useful as a screening marker for early predicting ofstomach cancer and the probability of contracting stomach cancer.

4. Conclusion

It is reported that the manifestation of the galectin-3 increases in themalignant tumor such as colon cancer, thyroid gland cancer, lung cancerand black epithelioma, and decreases in the breast cancer. In case ofstomach cancer, it is reported that the galectin-3 seldom manifest inthe normal tissue of stomach, but manifest in tissue of the stomachcancer [30]. In the above report, the manifestation of the galectin-3 innormal stomach tissue and stomach cancer was studied, but themanifestation of the galectin-3 during development of cancer was notestablished, thus the use of galectin-3 as an antigen for earlypredicting of tumor was not developed in the prior report. In contrast,the manifestations of the galectin-3 in normal tissue and blood, formerstage of stomach cancer and initial stage of benign tumor andcomplicated malignant tumor were studied in the present invention, andit is found that the over-manifestations of the galectin-3 can be usedas a signal for initiation of the malignant tumor, namely, a signal fordetecting the former and initial stages of the tumor development.

In this disclosure, there is shown and described only the preferredexamples of the invention, but, as aforementioned, it is to beunderstood that the invention is capable of use in various othercombinations and environments and is capable of changes or modificationswithin the scope of the inventive concepts as expressed herein.

REFERECES

-   1. Barondes, S. H., Cooper, D. N. W., Gift, M. A. and Leffler, H.:    Galectins structure and function of a large family of animal    lectins, J. Biol. Chem., 269 20807-20810, 1994.-   2. Barondes, S. H., Castronovo, V., Cooper, D. N. W., Cummings, R.    D., Drickamer, K., Feizi, T., Gitt, M. A., Hirabayashi, J., Hughes,    C., Kasai, K-I., Leffler, H., Liu, F-T., Lotan, R., Mercurio, A. M.,    Monsigny, M., Pillai, S., Poirer, F., Raz, A., Rigby, P. W. J.,    Rini, J. M. and Wang, J. L.: Galectins: a family of animal    β-galactoside-binding lectins, Cell, 76 : 597-598, 1994.-   3. Blaser, C., Kaufmann, M., Muller, C., Zimmermann, C., Wells, V.,    Mallucci, L. and Pircher, H.: β-galactoside-binding protein secreted    by activated T-cells inhibits antigen-induced proliferation of    T-cells, Eur. J. Immunol., 28 : 2311-2319, 1998.-   4. Bresalier, R. S., Yan, P-S., Byrd, J. C., Lotan, R. and Raz, A.    Expression of the endogenous galactose-binding protein galectin-3    correlates with the malignant potential of tumors in the central    nervous system, Cancer, 80 : 776-787,1997.-   5. Bresalier, R. S., Mazurek, N., Stemberg, L. R., Byrd, J. C.,    Yunker, C. K., Nangia-Makker, P. and Raz, A.: Metastasis of human    colon cancer is altered by modifyng expression of the    β-galactoside-binding protein galectin-3, Gastroenterology, 115 :    287-296, 1998.-   6. Castronovo, V., Van den Brule, F. A., Jackers, P., Clausse, N.,    Liu, F-T., Gillet, C. and Sobel, M. E.: Decreased expression of    galectin-3 is associated with progression of human breast cancer, J.    Pathol., 179 : 4348, 1996.-   7. Dagher, S. F., Wang, J. L and Patterson, R. J.: Identification of    galectin-3 as a factor in pre-mRNA splicing, Proc. Natl. Acad., 92:    1213-1217, 1995.-   8. Dong, S and Hughes, C.: Galectin-3 stimulates uptake of    extracellular Ca2+ in human JurkatT-cells, FEBS Letters, 395:    165-169,1996.-   9. Fernandez, P. L., Merino, M. J., Gomez, M., Campo, E., Medina,    T., Castronovo, V., Sanjuan, X., Cardesa, A., Liu, F-T. and    Sobel, M. E.: Galectin-3 and laminin expression in neoplastic and    non-neoplastic thyroid tissue, J. Pathol., 181 : 80-86,1997.-   10. Gitt, M. A., Wiser, M. F., Leffler, H., Herrmann, J., Xia, Y-R.,    Massa, S. M., Cooper, D. N. W., Lusis, A. J. and Barondes, S. H.:    Sequence and mapping of galectin-5, a β-galactoside-binding lectin,    found in rat erythrocytes, J. Biol. Chem., 270: 5032-5038, 1995.-   11. Hermanek, P and Sobin L. H.: TNM classification of malignant    tumors, 4th ed. Geneva: Union Internationale Contrele Cancer (UICC),    1987-   12. Ho, M-K. and Springer, T. A.: Mac-2, a novel 32,000 Mr mouse    macrophage subpopulation-specific antigen defined by monoclonal    antibodies, J. Immunol., 128:1221-1228,1982-   13. Hsu, D. K., Dowling, C. A., Jeng, K. C. G., Chen, J-T., Yang,    R-Y. and Liu, F-T.: Galectin-3 expression is induced in cirrhotic    liver and hepatocellular carcinoma, Int. J. Cancer, 81: 519-526,1999-   14. Irimura, T., Matsushida, Y., Sutton, R. C., Carralero, D.,    Ohannesian, D. W., Cleary, K. R., Ota, D. M., Nicolson, G. L. and    Lotan, R.: Increased content of an endogenous lactose-binding lectin    in human colorectal carcinoma progressed to metastatic stages,    Cancer Res., 51: 387-393, 1991-   15. Leffler, H. and Barondes, S. H.: Specificity of binding of three    soluble rat lung lectins to substituted and unsubstituted mammalian    α-galactosides, J. Biol. Chem.: 261: 10119-10126, 1986-   16. Lotan, R., Ito, H., Yasui, W., Yokozaki, H., Lotan, D. and    Tahara, E. Expression of a 31 kDa lactoside-binding lectin in normal    human gastric mucosa and in primary and metastatic gastric    carcinomas, Int. J. Cancer, 56 : 474-480, 1994-   17. Madsen, P., Rasmussen, H. H., Flint, T., Gromov, P., Kruse, T.    A., Honore, B., Vorum, H. and Celis, J. E.: Cloning, expression and    chromosome mapping of human galectin-7, J. Biol. Chem., 270:    5823-5829, 1995-   18. Marer, N. L. and Hughes, R. C.: Effects of the    carbohydrate-binding protein galectin-3 on the invasiveness of human    breast carcinoma cells, J. Cell. Physiol., 168: 51-58, 1996-   19. Ohannesian, D. W., Lotan, D., Thomas, P., Jeesup, J. M., Fukuda,    M., Gabius, H-J. and Lotan, R.: Carcinoembryonic antigen and other    glycoconjugates act as ligands for galectin-3 in human colon    carcinoma cells, Cancer Res., 55 2191-2199,1995-   20. Orlandi, F., Saggiorato, E., Pivano, G., Puligheddu, B.,    Termine, A., Cappia, S., Giuli, P. D. and Angeli, A.: Galectin-3 is    a presurgical marker of human thyroid carcinoma, Cancer Res., 58:    3015-3020, 1998-   21. Perrillo, N. L., Marcus, M. E. and Baum, L. G.: Galectins:    versatile modulators of cell adhesion cell proliferation and cell    death, J. Mol. Med., 76 : 402-412, 1998-   22. Raimond, J., Roulex, F., Monsigny, M. and Legrand, A.: The    second intron of the human galectin-3 gene has a strong promoter    activity down-regulated by p53, FEBS Letters, 363: 165-169, 1995-   23. Sanjuan, X., Fernandez, P. L., Castells, A., Castronovo, V., Van    Den Brule, F., Liu, F-T., Cardesa, A. and Campo, E.: Differential    expression of galectin-3 and galectin-1 in colorectal cancer    progression, Gastroenterology, 113: 1906-1915,1997-   24. Schoeppner, H. L., Raz, A., Ho, S. B. and Bresalier, R. S.:    Expression of an endogenous galactose-binding lectin correlates with    neoplastic progression in the colon, Cancer, 75: 2818-2826, 1995-   25. Sparrow, C. P., Leffler, H. and Barondes, S. H.: Multiple    soluble galactoside-binding lectins from human lung, J. Biol. Chem.,    262 : 7383-7390, 1987-   26. Woo, H-J., Lotz, M. M., Jung, J. U. and Mercurio, A. M.:    Carbohydrate-binding protein 35 (Mac-2), a laminin-binding lectin,    forms functional dimers using cysteine 186, J. Biol. Chem., 266:    18419-18422, 1991-   27. Woo, H-J., Shaw, L. M., Messier, J. M. and Mercurio. A. M.: The    major non-integrin laminin binding protein of macrophages is    identical to carbohydrate binding proein 35 (Mac-2), J. Biol. Chem.,    265: 7097-7099, 1990-   28. Xu, X-C., El-Naggar and Lotan, R.: Differential expression of    galectin-1 and galectin-3 in thyroid tumors, Am. J. Pathol., 147:    815-822, 1995-   29. Yang, R-Y., Hsu. D. K. and Liu, F-T.: Expression of galectin-3    modulates T-cell growth and apoptosis, Proc. Natl. Acad. Sci., 93 :    6737-3742, 1996-   30. Joo, H-G., and Woo, H-J.: Studies on the expression of    galectin-3 in gastric cancer, Kor. J. Immunol., 18: 583-590, 1996-   31. Woo, H-J.: Secretion of macrophage differentiation antigen,    Mac-2, Kor. J. Immunol., 15: 61-68, 1993.

1. A method of diagnosing or predicting the occurrence of a cancer orthe risk of contracting a cancer comprising the steps of: determining aconcentration of galectin-3 in a blood sample by reacting the bloodsample with a monoclonal antibody of the galectin-3; comparing thedetermined concentration of the galectin-3 with concentration of thegalectin-3 in a blood sample of a normal human; and predicting the riskof contracting a cancer if the determined concentration is greater thanthe concentration of the galectin-3 in blood of the normal human.
 2. Themethod according to claim 1, wherein the step of determining theconcentration of galectin-3 in the blood sample is carried out bymeasuring absorbance of the gallectin-3 and the monoclonal antibodycomplex, which is formed by enzyme-linked Immunosorbent assay (ELISA)method.
 3. The method according to claim 2, wherein the monoclonalantibody, which is a first antibody, is M3/38 monoclonal antibody (ratIgG), and a horse radish peroxidase (HRP)-conjugated goat anti-rat IgGis used as a second antibody which reacts with the first antibody. 4.The method according to claim 1, wherein the step of determining theconcentration of galectin-3 in the blood sample comprises the steps of:diluting the blood sample with a buffer solution to a predetermineddilution ratio; fixing the galectins-3 in the blood sample on astationary phase; reacting the monoclonal antibody of the galectin-3 asthe first antibody with the galectins-3 fixed on the stationary phase;reacting a horse radish peroxidase (HRP)-conjugated goat anti-rat IgG asthe second antibody with the first antibody; applying a colorantsolution to the horse radish peroxidase; and determining theconcentration of the galectins-3 by measuring an absorbance of thegalectin-3 and the antibody complex.
 5. The method according to claim 1,wherein the concentration of galectin-3 in the blood sample isdetermined while diluting an original blood sample with a dilution ratioof from 32 to
 256. 6. A method of diagnosing or predicting theoccurrence of cancer or the risk of contracting a cancer comprising thesteps of: forming an assay strip having a reaction part, a sampleinjection part, and a membrane for providing passage from the sampleinjection part to the reaction part, wherein a capture antibody ofgalectin-3 or galectin-3 is immobilized on the reaction part; moving ablood sample including galectin-3 and a gold-conjugated tracer antibodyof the galectin-3 trough the membrane from the sample injection part tothe reaction part; and predicting the risk of contracting a canceraccording to the color change of the reaction part.
 7. The methodaccording to claim 6, wherein the gold-conjugated tracer antibody isdirectly mixed with the blood sample and injected onto the sampleinjection part.
 8. The method according to claim 6, wherein the assaystrip further includes the gold-conjugated tracer antibody positionedbetween the sample injection part and the reaction part in a driedstate.
 9. The method according to claim 6, wherein the assay stripfurther includes a control line formed on the membrane at the oppositeend of the sample injection part, which is designed to change its colorwhen the blood sample arrives to the control line.
 10. The methodaccording to claim 6, wherein the blood sample is diluted with adilution ratio of from 32 to
 256. 11. A kit for diagnosing or predictingthe occurrence of cancer or the risk of contracting a cancer comprising:an assay strip having a reaction part; a sample injection part: and amembrane for providing sample passage from the sample injection part tothe reaction part, wherein the reaction part includes a capture antibodyof galectin-3 or galectin-3 immobilized thereon so that a color of thereaction part is determined according to the concentration of galectin-3in a blood sample when the blood sample including galectin-3 and agold-conjugated tracer antibody of the galectin-3 reaches from thesample injection part to the reaction part trough the membrane.
 12. Akit according to claim 11, wherein the gold-conjugated tracer antibodyis positioned on the assay strip between the sample injection part andthe reaction part in a dried state.
 13. A kit according to claim 11,wherein the assay strip further includes a control line formed on themembrane at the opposite end of the sample injection part, which isdesigned to change its color when the blood sample arrives to thecontrol line.
 14. A kit for diagnosing or predicting the occurrence ofcancer or the risk of contracting a cancer comprising: a microplate forimmobilizing galectin-3 in a blood sample; and a monoclonal antibody toreact with the galectin-3 immobilized on the microplate to induce acolor change of the microplate.
 15. A method of diagnosing or predictingthe occurrence of cancer or the risk of contracting a cancer comprisingthe steps of: determining a cancer screening antigen which manifests inthe stage of a adenoma or a chronic inflammation which are the formerstages of the malignant tumor development; determining a concentrationof the cancer screening antigen in a blood sample by reacting the bloodsample with a monoclonal antibody of the cancer screening antigen;comparing the determined concentration of the cancer screening antigenwith a concentration of the cancer screening antigen in a blood sampleof a normal human; and predicting the risk of contracting a cancer ifthe determined concentration is substantially greater or lesser than theconcentration of the cancer screening antigen in blood of the normalhuman.
 16. A method according to claim 16, wherein the step ofdetermining a cancer screening antigen comprises the steps of: measuringthe degree of the manifestation of an antigen in tissue at the stage ofnormal, chronic inflammation, adenoma, and malignant tumor; anddetermining the antigen as the cancer screening antigen when the degreeof the manifestation of the antigen in tissue at the stage of chronicinflammation or adenoma is substantially greater or lesser than thedegree of the manifestation of the antigen in tissue at the stage ofnormal.